Abcb4-defect cholangitis mouse model with hydrophobic bile acid composition by in vivo liver-specific gene deletion

Progressive familial intrahepatic cholestasis (PFIC) is a liver disease that occurs during childhood and requires liver transplantation. ABCB4 is localized along the canalicular membranes of hepatocytes, transports phosphatidylcholine into bile, and its mutation causes PFIC3. Abcb4 gene-deficient mice established as animal models of PFIC3 exhibit cholestasis-induced liver injury. However, their phenotypes are often milder than those of human PFIC3, partly because of the existence of large amounts of less toxic hydrophilic bile acids synthesized by the rodent-specific enzymes Cyp2c70 and Cyp2a12. Mice with double deletions of Cyp2c70/Cyp2a12 (CYPDKO mice) have a human-like hydrophobic bile acid composition. PFIC-related gene mutations were induced in CYPDKO mice to determine whether these triple-gene-deficient mice are a better model for PFIC. To establish a PFIC3 mouse model using CYPDKO mice, we induced abcb4 gene deletion in vivo using adeno-associated viruses expressing SaCas9 under the control of a liver-specific promoter and abcb4-target gRNAs. Compared to Abcb4-deficient wild-type mice, Abcb4-deficient CYPDKO mice showed more pronounced liver injury along with an elevation of inflammatory and fibrotic markers. The proliferation of intrahepatic bile ductal cells and hematopoietic cell infiltration were also observed. CYPDKO/abcb4-deficient mice show a predominance of taurine-conjugated chenodeoxycholic acid and lithocholic acid in the liver. In addition, phospholipid levels in the gallbladder bile were barely detectable. Mice with both human-like bile acid composition and Abcb4-defect exhibit severe cholestatic liver injury and are useful for studying human cholestatic diseases and developing new treatments.


Serum physiological marker analyses
After anesthesia, blood was collected from the hearts of the mice.Serum was separated using Bloodsepar (ImmunoBiological Laboratories Co., Ltd., Gunma, Japan).The levels of total cholesterol, HDL, triglycerides, AST, ALT, ALP, and total bilirubin were measured using Spotchem (Arkray.Inc, Kyoto, Japan).The ranges of measurements for each compound were as follows: total cholesterol 50-400 mg/dL; HDL, 10-150 mg/dL; triglycerides: 25-500 mg/dL; AST and ALT, 10-1000 IU/L; ALP, 38-113 IU/L; total bilirubin, 0.4-1.5 mg/dL.Values lower than the detection limit were defined as the lowest values.Serum total bile acid levels were analyzed using Total Bile Acid Assay Kit (CBL-STA631, Cell Biolabs.Inc.San diego, CA) according to the product manuscript.

Histological analysis
The livers were fixed in 4% paraformaldehyde (FUJIFILM Wako Pure Chemical) overnight and embedded in paraffin.Paraffin-embedded sections were analyzed by hematoxylin and eosin and Sirius red staining using standard protocols (1).After each staining, pathological specimens were observed, and images were captured using a BX63 microscope (Olympus, Tokyo, Japan).The degree of fibrosis was calculated using image sections stained with Sirius Red.Five representative images were collected from each mouse liver sections and the positive signals were quantified using Image J software (Bethesda, MA).
Immunohistochemical staining was performed to detect keratin 19 (K19) and CD45 in bile ductal and hematopoietic cells.Paraffin-embedded sections were heated at 110 °C for 10 min in target retrieval solution (Dako, Santa Clara, CA, USA).After blocking with 5% donkey serum/ PBS, the sections were incubated with either rat anti-CD45 (550539, BD Biosciences, Franklin Lakes, NJ, USA) or rabbit anti-K19 (provided by Prof. Miyajima, University of Tokyo) antibodies overnight at 4 °C.After washing, the sections were incubated with DyLight594 anti-rat IgG (Thermo Fisher Scientific) and Alexa488 anti-rabbit IgG (Thermo Fisher Scientific) for 60 min at room temperature.The fluorescence was observed using an Axio Imager M2 microscope.The expression of K19 and CD45 was calculated using image sections.Five representative images were collected from each mouse liver sections and the positive signals were quantified using Image J software.

Analyses of cholesterol, phospholipid and bile acid levels in the gallbladder
Mouse liver gallbladders were collected and biliary cholesterol and phospholipid concentration were determined using LabAssay Kits (FUJIFILM Wako Pure Chemical).Biliary total bile acid levels were analyzed using Total Bile Acid Assay Kit (CBL-STA631, Cell Biolabs.Inc.).

Analyses of bile acid composition in the liver
Liver samples were solubilized in 1M NaOH/water at 80 °C for 20 min; After the addition of internal standards and 0.5 mol/L potassium phosphate buffer (pH 7.4), bile acids were extracted and quantified by Liquid Chromatography-Mass Spectrometry.The mass analysis protocol has been previously described (2).The hydrophobicity indices were calculated using the individual bile acid data from the previous report (3).The hydrophobicity indices of individual bile acids not listed in the previous reports were obtained from our HPLC data and tentatively defined as ωMCA (-0.69), αMCA (-0.68), βMCA (-0.62),TωMCA (-0.85) and LCA (1.22).

Liver lipid purification and analyses.
Liver lipids (CYPDKO/NTC, n=7; CYPDKO/Abcb4-KO, n=5) were purified as previously described (4).The frozen liver tissues were weighed and homogenized in distilled water.Lipids were extracted from the homogenate with three volumes of 2:1 (v/v) chloroform and methanol.After centrifugation, the lower layers were collected and evaporated.The precipitated lipids were dissolved in isopropyl alcohol-Triton X-100 (9:1 v/v).The phospholipids and cholesterol in the extracts were analyzed using LabAssay Kits (FUJIFILM Wako Pure Chemical).

Figure
Figure S1 Liver-specific genome-editing analyses using mutant GFP-mice.(A) Analysis of

Figure
Figure S1 Liver-specific genome-editing analyses using mutant GFP-mice.(A) Analysis of genome-editing efficiency using mutant GFP-mice.AAVs expressing SaCas9 under liver-specific promoter and target gRNA against mutant GFP under human-U6 promoter were injected into 2-to 9-week-old mice.After 2-8-weeks of the injection, the organs were excised and recovery of GFP activity was confirmed by fluorescence microscopy.(B) Schematic diagram of the liver-specific SaCas9-expressing AAV vectors.(C) Comparison of genome-editing efficiency of liver-specific SaCas9-expressing AAV vectors.Mutant GFP-mice (9-week-old) were infected with AAV and livers were analyzed with a fluorescence microscope after 4 weeks of infection.(D and E) Comparison of genome-editing efficiency in the liver and other tissues.Liver-specific genome-editing in juvenile (2-weeksold) mice was induced by the injection of AAV.After 8 weeks (D) and 2 weeks (E), recovered GFP activities in the liver and other tissues were analyzed.White line, 100 µm.

Table S1
LCA free 12oxo-CDCA nmol/whole liver nmol/whole liver nmol/whole liver nmol/whole liver nmol/whole liver nmol/whole liver nmol/whole liver nmol/whole liver nmol/whole liver nmol/whole liver nmol/whole liver nmol/whole liver nmol/whole liver nmol/whole liver liver nmol/whole liver nmol/whole liver nmol/whole liver nmol/whole liver nmol/whole liver nmol/whole liver nmol/whole liver nmol/whole liver nmol/whole liver nmol/whole liver nmol/whole liver nmol/whole liver nmol/whole liver